[CANCER RESEARCH 60, 1604 –1608, March 15, 2000]
Peroxisome Proliferator-activated Receptor ␥ Ligands Inhibit Estrogen Biosynthesis
in Human Breast Adipose Tissue: Possible Implications for Breast Cancer Therapy1
Gary L. Rubin,2 Ying Zhao, Allan M. Kalus, and Evan R. Simpson
Victorian Breast Cancer Research Consortium, Inc., Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia [G. L. R., E. R. S.]; Department of Radiology,
University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051 [Y. Z.]; and The Plastic & Aesthetic Surgery Centre, Windsor, Victoria 3181, Australia [A. M. K.]
ABSTRACT
Estrogen biosynthesis is catalyzed by aromatase cytochrome P-450 (the
product of the CYP19 gene). Adipose tissue is the major site of estrogen
biosynthesis in postmenopausal women, with the local production of estrogen in breast adipose tissue implicated in the development of breast
cancer. In human adipose tissue, aromatase is primarily expressed in the
mesenchymal stromal cells and is a marker of the undifferentiated preadipocyte phenotype. Aromatase expression in adipose tissue is regulated
via the distal promoter I.4, under the control of glucocorticoids and class
I cytokines such as oncostatin M, interleukin 6, and interleukin 11, as well
as tumor necrosis factor ␣. These cytokines, which are expressed in
adipose, also inhibit adipocyte differentiation. Therefore, we hypothesized
that factors which stimulate adipocyte differentiation should inhibit aromatase expression. These factors include synthetic peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands such as thiazolidinediones, e.g.,
troglitazone and rosiglitazone (BRL49653) and the endogenous PPAR␥
ligand 15-deoxy-⌬12,14-prostaglandin J2. We have demonstrated by measurement of aromatase activity and by reverse transcription-PCR/Southern blotting that these PPAR␥ ligands inhibit aromatase expression in
cultured breast adipose stromal cells stimulated with oncostatin M or
tumor necrosis factor ␣ plus dexamethasone in a concentration-dependent
manner, whereas a metabolite of troglitazone that does not activate
PPAR␥ has no effect. We have also shown that troglitazone inhibits
luciferase activity of reporter constructs containing various lengths of the
upstream region of promoter I.4 transfected into mouse 3T3-L1 preadipocyte mesenchymal cells, whereas the troglitazone metabolite does not.
Because local estrogen production in breast fat is implicated in breast
cancer development in postmenopausal women, the actions of PPAR␥
ligands suggest that they may have potential therapeutic benefit in the
treatment and management of breast cancer.
INTRODUCTION
Estrogen biosynthesis is catalyzed by the enzyme aromatase (Refs.
1– 4; aromatase cytochrome P-450; the product of the CYP19 gene;
Ref. 5). CYP19 is a member of the P-450 superfamily of genes, which
currently contains over 600 members in some 40 gene families (6).
Aromatase is responsible for catalyzing the aromatization of the A
ring of C19 androgens to the phenolic A ring of C18 estrogens,
resulting in loss of the C19 angular methyl group as formic acid (7).
In humans, aromatase is expressed in a variety of tissues including:
the granulosa cells and corpus luteum of the ovary (8, 9); the Leydig
cells and germ cells of the testis (10, 11); the syncytiotrophoblast of
the placenta (8); various sites in the brain including the hypothalamus
and hippocampus (12, 13); adipose tissue of the breast, abdomen,
thighs, and buttocks (14, 15); and osteoblasts of bone (16, 17). The
human CYP19 gene spans at least 75 kb, with a coding region of ⬃35
kb containing nine translated exons (II-X; Refs. 18 –20; Fig. 1).
Aromatase transcripts in the various tissue sites of expression contain
different 5⬘-untranslated first exons because of the use of a number of
alternative promoters that regulate aromatase expression in the ovary
Received 8/30/99; accepted 1/18/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by a grant from The Victorian Breast Cancer Research Consortium, Inc.
(Anti-Cancer Council of Victoria, Australia) and by USPHS Grant R37-AG08174.
2
To whom requests for reprints should be addressed, at Prince Henry’s Institute of
Medical Research, P. O. Box 5152, Clayton, Victoria 3168, Australia. Phone: 61-3-95943570; Fax: 61-3-9594-6125; E-mail: [email protected].
(promoter II; Ref. 9), placenta (promoter I.1; Refs. 8, 21, and 22), and
adipose tissue (promoters I.4 and II; Ref. 23) via alternative splicing
mechanisms. Each promoter is differentially regulated with promoter
II under the control of follicle-stimulating hormone, the actions of
which are mediated by cyclic AMP (24, 25), whereas promoter I.1 is
regulated by retinoids (26). Expression via promoter I.4 in adipose
tissue requires the synergistic actions of glucocorticoids and class I
cytokines or TNF3-␣ (27–29). The former uses a Janus-activated
kinase 1/STAT3 signaling pathway in which the activated STAT3
binds to a GAS element located upstream of promoter I.4 (28),
whereas the latter uses a mitogen-activated protein kinase/AP-1 pathway, resulting in binding of c-fos/c-jun to an AP-1 site upstream of the
GAS element (Ref. 29; Fig. 1).
Adipose tissue is the main site of estrogen biosynthesis in postmenopausal women, and aromatase expression in adipose increases
with age and body weight (14, 15). Local production of estrogen in
breast adipose tissue is implicated in the development of breast cancer
in postmenopausal women (15, 30). Estrogen levels in breast tumors
are as much as 10 times greater than in the circulation of postmenopausal women (31). This appears to be because aromatase expression
within a tumor and the surrounding breast tissue is elevated as a result
of the production of factors by the tumor which stimulate aromatase
expression (32). Aromatase expression in adipose tissue is primarily
located in the mesenchymal stromal cells and appears to be a marker
of the undifferentiated preadipocyte phenotype (33–35). Consistent
with this, factors known to stimulate aromatase expression in adipose
tissue, such as the class I cytokines interleukin 6, interleukin 11,
OSM, as well as TNF-␣, also inhibit adipocyte differentiation (36,
37). Conversely, factors that stimulate adipocyte differentiation would
be anticipated to inhibit aromatase expression. Such factors include
ligands of PPAR␥, such as the TZDs troglitazone and rosiglitazone, as
well as natural ligands such as 15d-PGJ2 (38, 39). PPAR␥ is a member
of the superfamily of ligand-activated transcription factors, which includes receptors for steroid, retinoid, and thyroid hormones, and exists in
three different isoforms, ␥1, ␥2, and ␥3. It is abundantly expressed in
adipose tissue and plays a key role in adipocyte differentiation (38 – 40).
In the present report, we show that TZDs and 15d-PGJ2 do indeed
inhibit aromatase expression in human adipose stromal cells of breast
origin, whereas a metabolite of troglitazone that does not activate
PPAR␥ has no action on aromatase expression. These findings suggest that troglitazone and other TZDs might have therapeutic utility in
the management of breast cancer in postmenopausal women.
MATERIALS AND METHODS
Cell Culture. Adipose tissue was obtained from women undergoing reduction mammoplasty or reduction abdominoplasty, after receiving informed
consent. Adipose stromal cells were isolated by collagenase digestion of
adipose tissue as described (33) and maintained in primary culture at 50,000
cells/ml in DMEM enriched medium (Trace Biosciences, Sydney, New South
Wales, Australia) supplemented with 15% FBS (15% v/v; Trace Biosciences)
and allowed to grow until confluent (5– 6 days) prior to treatment. After serum
3
The abbreviations used are: TNF, tumor necrosis factor; STAT, signal transducers
and activators of transcription; GAS, IFN-␥ activating sequence; OSM, oncostatin M;
PPAR␥, peroxisome proliferator-activated receptor ␥; TZD, thiazolidinedione; 15d-PGJ2,
15-deoxy-⌬12,14-prostaglandin J2; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Dex, dexamethasone; Luc, luciferase; AP-1, activating protein-1.
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PPAR␥ LIGANDS INHIBIT AROMATASE IN BREAST ADIPOSE
Fig. 1. The structure of the human CYP19 gene. The genomic region
spans at least 75 kb, with a coding region of ⬃35 kb containing nine
translated exons (II–X), represented by the closed bars. The heme
binding region (HBR) is located in exon X, as are two alternative
polyadenylation signals that give rise to two different aromatase transcripts of 3.4 and 2.9 kb. The 5⬘-region of the CYP19 gene contains
several untranslated exons, e.g., I.1, I.3, and I.4, represented by the open
bars. Expression of aromatase is controlled in a tissue-specific manner
via the use of these alternative promoters. Because the various first exons
are spliced into a common 3⬘-splice junction upstream of the start of
translation, the coding region and hence the protein product are identical
in each tissue site of expression. The identified regulatory elements,
AP-1, GAS, and GRE (glucocorticoid response element) upstream of
promoter I.4, are indicated. Arom, aromatase.
deprivation for 24 h, cells were treated for another 24 h with 250 nM dexamethasone and 5 ng/ml of recombinant human OSM (Sigma, Sydney, New
South Wales, Australia) in the presence or absence of the PPAR␥ ligands
troglitazone (Parke-Davis, Ann Arbor, MI), rosiglitazone (BRL49653), or
15d-PGJ2 (Cayman Chemical Co., Ann Arbor, MI) at varying concentrations.
Cells were either assayed for aromatase activity or harvested for total RNA.
Aromatase Assay. Aromatase activity was determined after incubation of
cells with [1␤-3H]androstenedione (NEN Life Science Products, Inc., Boston,
MA) for 2 h and measured by the incorporation of tritium into [3H]water
(NEN), as described previously (33).
RT-PCR and Southern Blot Analysis. After treatment, total RNA was
extracted from human breast adipose stromal cells using the RNeasy Mini kit
(Qiagen, Melbourne, Victoria, Australia), according to the manufacturer’s
instructions. Total RNA (0.25 ␮g) was reverse transcribed using the Superscript One-Step RT-PCR system (Life Technologies, Inc., Melbourne, Victoria, Australia) as described by the manufacturer, using the aromatase-specific
primers (Pacific Oligos, Sydney, New South Wales, QLD, Australia): 5⬘-sense
from exon I.4 (5⬘-GTG ACC AAC TGG AGC CTG-3⬘); 5⬘-sense from exon
II (5⬘-TTG GAA ATG CTG AAC CCG-3⬘); and 3⬘-antisense from exon III
(5⬘-CAG GAA TCT GCC GTG GGA GA-3⬘). Product amplification was
determined as a function of the number of cycles to derive the linear range. On
the basis of this information, amplification was routinely continued for 30
cycles. Integrity of cDNA was checked by amplification of the GAPDH gene
using the GAPDH-specific primers (Pacific Oligos, Sydney, New South
Wales): 5⬘-sense (5⬘-CGG AGT CAA CGG ATT TGG TCG TAT-3⬘) and
3⬘-antisense (5⬘-AGC CTT CTC CAT GGT GGT GAA GAC-3⬘). The PCR
products were subjected to electrophoresis and blotted onto Hybond-N⫹
(Amersham Pharmacia Biotech, Sydney, New South Wales, Australia; Ref.
41). For quantitation, membranes were hybridized with specific 32P-labeled
oligoprobes to aromatase exon II (5⬘-CAT CAC CAG CAT CGT GCC TG-3⬘)
and GAPDH (5⬘-AAG ATG GTG ATG GGA TTT CC-3⬘) labeled by T4
polynucleotide kinase (Life Technologies) and [␥-32P]ATP (NEN). Aromatase
expression was quantitated (arbitrary units) by phosphorimaging (Bio-imaging
analyzer MacBAS v2.4; Fujifilm) and normalized against GAPDH.
Transient Transfection and Reporter Gene Assays. 3T3-L1 cells, a
mouse preadipocyte fibroblast cell line (ATCC CL-173) were cultured in
DMEM supplemented with 10% FBS at a density of 40,000 cells/ml. 3T3-L1
cells were transfected for 22 h with 1.5 ␮g of a chimeric DNA reporter gene
construct containing 774 bp of the 5⬘-regulatory region upstream of promoter
I.4 of the human CYP19 gene fused to a Luc reporter gene in the pGL3 vector
(a generous gift from Dr. Makio Shozu, Kanazawa University, Kanazawa,
Japan) and 0.5 ␮g of pSV-␤-galactosidase control vector (Promega, Sydney,
New South Wales, Australia) using FuGENE 6 Transfection Reagent (Roche
Diagnostics Australia Pty. Ltd., Victoria, Melbourne, Australia) according to
manufacturer’s instructions. After serum starvation, cells were stimulated with
Dex and OSM for 8 h and treated in the presence or absence of troglitazone or
troglitazone metabolite. Cells were then lysed, and relative Luc activity was
measured and normalized to ␤-galactosidase activity (Promega).
RESULTS
PPAR␥ Ligands Inhibit Aromatase Activity in Human Breast
Adipose Stromal Cells. The effect of PPAR␥ ligands on aromatase
activity in human breast adipose stromal cells was determined. Basal
levels of aromatase activity in these cells are low but can be markedly
up-regulated in the presence of both glucocorticoids and class I
cytokines or TNF-␣ (28, 29). The PPAR␥ ligands troglitazone, rosiglitazone (BRL49653), and 15d-PGJ2 inhibited Dex- and OSMstimulated aromatase activity in human breast adipose stromal cells in
a concentration-dependent manner, whereas a troglitazone metabolite
that was not a PPAR␥ ligand had no effect (Fig. 2A). Similar results
were obtained when TNF-␣ was substituted for OSM (Fig. 2B). The
endogenous PPAR␥ ligand 15d-PGJ2 was the most potent inhibitor of
aromatase activity, with an IC50 of ⬍1 ␮M. Concentrations of 15dPGJ2 ⬎2 ␮M completely abolished aromatase activity, whereas concentrations ⬍100 nM had little effect. The synthetic PPAR␥ ligand
troglitazone inhibited aromatase activity with an IC50 of ⬍10 ␮M.
Concentrations of troglitazone ⬎10 ␮M completely abolished aro-
Fig. 2. PPAR␥ ligands inhibit aromatase activity in human breast adipose stromal cells.
Human breast adipose stromal cells were treated for 24 h with 250 nM Dex and 5 ng/ml
OSM (A) or 5 ng/ml TNF-␣ (B) in the presence or absence of various PPAR␥ ligands:
troglitazone, 15d-PGJ2, BRL49653 (rosiglitazone), or a troglitazone metabolite. Aromatase activity (pmol/mg protein/2 h) was determined after a 2-h incubation with the
aromatase substrate [1␤-3H]androstenedione and expressed as a percentage of the positive
control (Dex/OSM or Dex/TNF␣). Each treatment was performed in triplicate and each
experiment repeated three times; bars, SE.
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PPAR␥ LIGANDS INHIBIT AROMATASE IN BREAST ADIPOSE
Fig. 3. PPAR␥ ligands inhibit aromatase expression in human breast adipose stromal cells. Human
breast adipose stromal cells were treated for 24 h
with 250 nM Dex and 5 ng/ml OSM in the presence
or absence of troglitazone (A and C) or 15d-PGJ2 (B
and D). Total RNA was extracted, and 0.25 ␮g of
total RNA was used for RT-PCR amplification of
aromatase promoter I.4-specific transcripts (A and B)
or coding region (C and D) for 30 cycles (D, 35
cycles). Integrity of cDNA was checked by amplification of GAPDH. Southern analysis was performed
using a 32P-labeled probe to aromatase or GAPDH.
Aromatase expression was quantitated by phosphorimaging and normalized against GAPDH.
matase activity. To demonstrate that this was not an action of troglitazone to inhibit the catalytic activity of the aromatase enzyme directly, a time course of the inhibitory action of troglitazone was
determined (data not shown). Troglitazone only blocked the induction
of aromatase activity by Dex and OSM after an incubation of 24 h or
greater. Troglitazone had no effect on aromatase activity when it was
added simultaneously with the aromatase substrate [1␤-3H]androstenedione.
PPAR␥ Ligands Inhibit Aromatase Expression in Human
Breast Adipose Stromal Cells. Troglitazone and 15d-PGJ2 also inhibit aromatase expression in a concentration-dependent manner, as
determined by RT-PCR amplification of promoter I.4-specific transcripts (Fig. 3, A and B). Aromatase expression in breast adipose
stromal cells is very low but is markedly stimulated by both Dex and
OSM. Treatment of Dex/OSM-stimulated adipose stromal cells with
troglitazone indicated that aromatase expression via promoter I.4 is
inhibited in a concentration-dependent manner and is almost completely abolished at concentrations ⬎10 ␮M (Fig. 3A). 15d-PGJ2 also
inhibited Dex/OSM-stimulated aromatase expression in a concentration-dependent manner and dramatically reduced aromatase expression at ⬎2 ␮M (Fig. 3B). No effect on the expression level of the
housekeeping gene GAPDH was observed with either ligand. Both
PPAR␥ ligands inhibited aromatase expression in a similar concentration range to that observed for inhibition of aromatase activity.
Similar results were obtained when the coding region of aromatase
was amplified using the 5⬘-primer to exon II (Fig. 3, C and D).
Troglitazone Inhibits Aromatase Transcription via the CYP19
Gene Promoter I.4. To gain further insight into how PPAR␥ ligands
interfere with the transcription of the aromatase gene via promoter
I.4, 3T3-L1 cells were transfected with either a Luc reporter gene
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PPAR␥ LIGANDS INHIBIT AROMATASE IN BREAST ADIPOSE
fused to ⫺774 bp of the 5⬘-regulatory region of promoter I.4 of the
CYP19 gene in the pGL3 vector or else the pGL3-basic vector. Fig. 4
demonstrates that in the absence of treatment, basal levels of Luc gene
expression were observed in cells transfected with both pGL3-basic
vector and ⫺774/⫹14 P450-I.4/Luc. Treatment with Dex and OSM
(which up-regulate aromatase expression through promoter I.4)
caused a 5-fold induction in Luc gene expression. Troglitazone caused
a concentration-dependent inhibition of Luc gene expression with
concentrations ⬎10 ␮M, reducing expression to basal levels. This
result was consistent with the effect of troglitazone on aromatase
activity (Fig. 2) and expression (Fig. 3). Treatment with troglitazone
had no effect on basal Luc or ␤-galactosidase expression, and treatment with a 0.1% DMSO vehicle did not affect Luc activity.
Troglitazone Inhibits Aromatase Transcription via Promoter
I.4 through Interaction with PPAR␥. To determine whether the
effect of troglitazone is mediated through its binding to PPAR␥,
3T3-L1 cells were transfected with the same Luc reporter gene constructs, ⫺774/⫹14 P450-I.4/Luc or pGL3-basic vector, and treated
with troglitazone and a troglitazone metabolite that is unable to bind
to the receptor. Troglitazone inhibited transcription via promoter I.4 in
a concentration-dependent manner as before, whereas the troglitazone
metabolite had no effect on gene expression at any concentration
(Fig. 5), similar to the results obtained in terms of aromatase
activity (Fig. 2).
DISCUSSION
PPAR␥ is a key transcription factor involved in adipocyte differentiation (38, 39). Because of their ability to increase the responsiveness of insulin-sensitive cells to insulin, ligands for PPAR␥ have been
actively studied for their therapeutic utility in the treatment of insulinresistant diabetes, and currently one of these, namely troglitazone, is
in clinical use in the United States for this purpose. More recently it
has been found that PPAR␥ ligands can stimulate differentiation of a
number of cell types and not only those of mesenchymal origin, e.g.,
colon cancer cells (42) as well as breast cancer cell lines (43),
suggesting that PPAR␥ ligands could have therapeutic utility in the
treatment of certain forms of cancer. Indeed, in a recent clinical trial
of patients with advanced liposarcoma, it was found that troglitazone
Fig. 4. Troglitazone inhibits aromatase transcription via promoter I.4. 3T3-L1 cells
were transfected for 22 h with 1.5 ␮g of a Luc reporter gene construct containing 774 bp
of the 5⬘-regulatory region upstream of the CYP19 gene promoter I.4 (⫺774/⫹14
P450-I.4/Luc) or pGL3-basic vector (pGL3b) and cotransfected with 0.5 ␮g of pSV-␤galactosidase control vector. Cells were stimulated for 8 h with 250 nM Dex and 5 ng/ml
OSM in the presence or absence of troglitazone (0.1% DMSO). Cells were lysed, and Luc
activity was determined and normalized to ␤-galactosidase activity. Bars, SE.
Fig. 5. Troglitazone inhibits aromatase transcription via promoter I.4 through PPAR␥.
3T3-L1 cells were transfected for 22 h with 1.5 ␮g of a Luc reporter gene construct
containing 774 bp of the 5⬘-regulatory region upstream of the CYP19 gene promoter I.4
(⫺774/⫹14 P450-I.4/Luc) or pGL3-basic vector (pGL3b) and cotransfected with 0.5 ␮g
of pSV-␤-galactosidase control vector. Cells were stimulated for 8 h with 250 nM Dex and
5 ng/ml OSM in the presence or absence of troglitazone or a troglitazone metabolite. Cells
were lysed, and Luc activity was determined and normalized to ␤-galactosidase activity.
Bars, SE.
can stimulate the differentiation of such tumors in vivo (44). The
observation that PPAR␥ ligands can stimulate the differentiation of
breast cancer cell lines (43) suggests that these compounds might have
utility in breast cancer therapy.
The results presented here provide independent data in support of
this contention, i.e., that PPAR␥ ligands inhibit the expression of
aromatase and hence estrogen biosynthesis in adipose tissue, and in
particular adipose tissue of the human breast. Aromatase expression is
a marker of the undifferentiated preadipocyte mesenchymal phenotype (33–35) and as such is stimulated by factors that inhibit adipocyte
differentiation, such as class I cytokines and TNF-␣, a number of
which are synthesized in adipose tissue itself (36, 37). Consequently,
it was anticipated that factors that stimulate adipocyte differentiation
and increase the expression of differentiation markers, such as lipoprotein lipase and the insulin receptor, would also inhibit aromatase
expression in adipose mesenchymal cells. The results presented here
show that this is indeed the case; aromatase expression stimulated by
class I cytokines or TNF-␣ is inhibited by several PPAR␥ ligands in
a concentration-dependent fashion, whereas a troglitazone metabolite,
which is not a PPAR␥ ligand, had no effect on aromatase expression.
The results of transfection experiments using chimeric constructs in
which the Luc reporter gene is regulated by the aromatase promoter
I.4 indicate that this inhibition is a consequence of interaction with the
cell signaling pathways involving Janus-activated kinase 1 and
STAT3 in the case of the class I cytokines, and AP-1 in the case of
TNF␣. Interactions of PPAR␥ with these signaling pathways have
previously been described in other systems, for example in a monocyte cell lineage, PPAR␥ interferes with IFN-␥ action via a STAT1and AP-1-mediated pathway (45). The mechanism whereby PPAR␥
inhibits cytokine-stimulated aromatase expression via promoter I.4 in
adipose stromal cells remains to be elucidated. One possibility is that
it competes with the STAT3 and AP-1 pathways for CREB-binding
protein, which appears to play a universal role in mediating the
transcriptional responses of genes to multiple signaling pathways.
This possibility is currently under investigation.
The results presented here suggest that PPAR␥ ligands could find
utility in breast cancer therapy. Presently, most breast cancer hormonal therapies are directed to inhibition of estrogen action or inhibition
of aromatase activity. Estrogen receptor antagonists such as tamoxifen
display selectivity in the tissue site of action, but generally after
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PPAR␥ LIGANDS INHIBIT AROMATASE IN BREAST ADIPOSE
several years of treatment, breakthrough occurs with clonal tumor
lines developing that are unresponsive to tamoxifen. Aromatase inhibitors are used as second-line therapy and also have potential as
first-line adjuvant therapy but have the disadvantage that they inhibit
aromatase indiscriminately in all tissue sites including bone and brain,
where they may have adverse effects in terms of bone mineralization
on the one hand and possibly cognitive function on the other. Because
aromatase expression is regulated differently in different tissue sites
through the use of different tissue-specific promoters (35), the possibility is presented that tissue-selective inhibitors of aromatase expression could be developed. Whether PPAR␥ ligands will demonstrate
sufficient tissue selectivity to warrant their development as breast
cancer therapeutic agents remains to be determined, but results presented here are sufficiently encouraging to warrant further investigation.
ACKNOWLEDGMENTS
We thank Parke-Davis, Ann Arbor, MI, for generous gifts of troglitazone
and the troglitazone metabolite. We also thank Sue Panckridge for skilled
graphic assistance.
REFERENCES
1. Thompson, E. A., Jr., and Siiteri, P. K. The involvement of human placental
microsomal cytochrome P450 in aromatization. J. Biol. Chem., 249: 5373–5378,
1974.
2. Mendelson, C. R., Wright, E. E., Evans, C. T., Porter, J. C., and Simpson, E. R.
Preparation and characterization of polyclonal and monoclonal antibodies against
human aromatase cytochrome P450 (P450arom), and their use in its purification.
Arch. Biochem. Biophys., 243: 480 – 491, 1985.
3. Nakajin, S., Shimoda, M., and Hall, P. F. Purification to homogeneity of aromatase
from human placenta. Biochem. Biophys. Res. Commun., 134: 704 –710, 1986.
4. Kellis, J. T., Jr., and Vickery, L. E. Purification and characterization of human
placental aromatase cytochrome P450. J. Biol. Chem., 262: 4413– 4420, 1987.
5. Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R. W.,
Feyereisen, R., Gonzalez, F. J., Coon, M. J., Gunsalus, I. C., Gotoh, O., Okuda, K.,
and Nebert, D. W. The P450 superfamily: update on new sequences, gene mapping,
accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell
Biol., 12: 1–51, 1993.
6. Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman,
D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C.,
and Nebert, D. W. P450 superfamily: update on new sequences, gene mapping,
accession numbers and nomenclature. Pharmacogenetics, 1: 1– 42, 1996.
7. Thompson, E. A., Jr., and Siiteri, P. K. Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J. Biol. Chem., 249: 5364 –5372, 1974.
8. Means, G. D., Kilgore, M. W., Mahendroo, M. S., Mendelson, C. R., and Simpson,
E. R. Tissue-specific promoters regulate aromatase cytochrome P450 gene expression
in human ovary and fetal tissues. Mol. Endocrinol., 5: 2005–2013, 1991.
9. Jenkins, C., Michael, D., Mahendroo, M., and Simpson, E. Exon-specific Northern
analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal
promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression
in human ovary. Mol. Cell. Endocrinol., 97: 1– 6, 1993.
10. Tsai-Morris, C. H., Aquilana, D. R., and Dufau, M. L. Cellular localization of rat
testicular aromatase activity during development. Endocrinology, 116: 38 – 46, 1985.
11. Nitta, H., Bunick, D., Hess, R. A., Janulis, L., Newton, S. C., Millette, C. F., Osawa,
Y., Shizuta, Y., Toda, K., and Bahr, J. M. Germ cells of the mouse testis express P450
aromatase. Endocrinology, 132: 1396 –1401, 1993.
12. Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., Kuhn, M.,
White, R. J., Takaoka, Y., and Wolin, L. The formation of estrogens by central
neuroendocrine tissues. Recent Prog. Horm. Res., 31: 295–319, 1975.
13. Roselli, C. E., Horton, L. E., and Resko, J. A. Distribution and regulation of
aromatase activity in the rat hypothalamus and limbic system. Endocrinology, 117:
2471–2474, 1985.
14. Grodin, J. M., Siiteri, P. K., and MacDonald, P. C. Source of estrogen production in
postmenopausal women. J. Clin. Endocrinol. Metab., 38: 207–214, 1973.
15. Bulun, S. E., and Simpson, E. R. Competitive reverse transcription-polymerase chain
reaction analysis indicates that levels of aromatase cytochrome P450 transcripts in
adipose tissue of buttocks, thighs, and abdomen of women increase with advancing
age. J. Clin. Endocrinol. Metab., 78: 428 – 432, 1994.
16. Bruch, H. R., Wolf, L., Budde, R., Romalo, G., and Schweikert, H. U. Androstenedione metabolism in cultured human osteoblast-like cells. J. Clin. Endocrinol. Metab.,
75: 101–105, 1992.
17. Shozu, M., and Simpson, E. R. Aromatase expression of human osteoblast-like cells.
Mol. Cell. Endocrinol., 139: 117–129, 1998.
18. Means, G. D., Mahendroo, M. S., Corbin, C. J., Mathis, J. M., Powell, F. E.,
Mendelson, C. R., and Simpson, E. R. Structural analysis of the gene encoding human
aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis.
J. Biol. Chem., 264: 19385–19391, 1989.
19. Harada, N., Yamada, K., Saito, K., Kibe, N., Dohmae, S., and Takagi, Y. Structural
characterization of the human estrogen synthetase (aromatase) gene. Biochem. Biophys. Res. Commun., 166: 365–372, 1990.
20. Toda, K., Terashima, M., Kamamoto, T., Sumimoto, H., Yamamoto, Y., Sagara, Y.,
Ikeda, H., and Shizuta, Y. Structural and functional characterization of human
aromatase P450 gene. Eur. J. Biochem., 193: 559 –565. 1990.
21. Mahendroo, M. S., Means, G. D., Mendelson, C. R., and Simpson, E. R. Tissuespecific expression of human P450arom. The promoter responsible for expression in
adipose tissue is different from that utilized in placenta. J. Biol. Chem., 266:
11276 –11281, 1991.
22. Kilgore, M. W., Means, G. D., Mendelson, C. R., and Simpson, E. R. Alternative
promotion of aromatase P-450 expression in the human placenta. Mol. Cell. Endocrinol., 83: 9 –16, 1992.
23. Mahendroo, M. S., Mendelson, C. R., and Simpson, E. R. Tissue-specific and
hormonally controlled alternative promoters regulate aromatase cytochrome P450
gene expression in human adipose tissue. J. Biol. Chem., 268: 19463–19470, 1993.
24. Michael, M. D., Kilgore, M. W., Morohashi, K., and Simpson, E. R. Ad4BP/SF-1
regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the
human aromatase P450 (CYP19) gene in the ovary. J. Biol. Chem., 270: 13561–
13566, 1995.
25. Michael, M. D., Michael, L. F., and Simpson, E. R. A CRE-like sequence that binds
CREB and contributes to cAMP-dependent regulation of the proximal promoter of the
human aromatase P450 (CYP19) gene. Mol. Cell. Endocrinol., 134: 147–156, 1997.
26. Sun, T., Zhao, Y., Mangelsdorf, D. J., and Simpson, E. R. Characterization of a region
upstream of exon I.1 of the human CYP19 (aromatase) gene that mediates regulation
by retinoids in human choriocarcinoma cells. Endocrinology, 139: 1684 –1691, 1998.
27. Zhao, Y., Mendelson, C. R., and Simpson, E. R. Characterization of the sequences of
the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in
adipose stromal cells and fetal hepatocytes. Mol. Endocrinol., 9: 340 –349, 1995.
28. Zhao, Y., Nichols, J. E., Bulun, S. E., Mendelson, C. R., and Simpson, E. R.
Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT
pathway in regulation of the adipose-specific promoter. J. Biol. Chem., 270: 16449 –
16457, 1995.
29. Zhao, Y., Nichols, J. E., Valdez, R., Mendelson, C. R., and Simpson, E. R. Tumor
necrosis factor-␣ stimulates aromatase gene expression in human adipose stromal
cells through use of an activating protein-1 binding site upstream of promoter 1.4.
Mol. Endocrinol., 10: 1350 –1357, 1996.
30. Bulun, S. E., Price, T. M., Aitken, J., Mahendroo, M. S., and Simpson, E. R. A link
between breast cancer and local estrogen biosynthesis suggested by quantification of
breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription [published erratum appears in
J. Clin. Endocrinol. Metab., 78: 494, 1994]. J. Clin. Endocrinol. Metab., 77: 1622–
1628, 1993.
31. van Landeghem, A. A., Poortman, J., Nabuurs, M., and Thijssen, J. H. Endogenous
concentration and subcellular distribution of estrogens in normal and malignant
human breast tissue. Cancer Res., 45: 2900 –2906, 1985.
32. Bulun, S. E., Mahendroo, M. S., and Simpson, E. R. Aromatase gene expression in
adipose tissue: relationship to breast cancer. J. Steroid Biochem. Mol. Biol., 49:
319 –326, 1994.
33. Ackerman, G. E., Smith, M. E., Mendelson, C. R., MacDonald, P. C., and Simpson,
E. R. Aromatization of androstenedione by human adipose tissue stromal cells in
monolayer culture. J. Clin. Endocrinol. Metab., 53: 412– 417, 1981.
34. Price, T., Aitken, J., Head, J., Mahendroo, M., Means, G., and Simpson, E. Determination of aromatase cytochrome P450 messenger ribonucleic acid in human breast
tissue by competitive polymerase chain reaction amplification. J. Clin. Endocrinol.
Metab., 74: 1247–1252, 1992.
35. Simpson, E. R., Zhao, Y., Agarwal, V. R., Michael, M. D., Bulun, S. E., Hinshelwood, M. M., Graham-Lorence, S., Sun, T., Fisher, C. R., Qin, K., and Mendelson,
C. R. Aromatase expression in health and disease. Recent Prog. Horm. Res., 52:
185–213, 1997.
36. Torti, F. M., Torti, S. V., Larrick, J. W., and Ringold, G. M. Modulation of adipocyte
differentiation by tumor necrosis factor and transforming growth factor ␤. J. Cell.
Biol., 108: 1105–1113, 1989.
37. Keller, D. C., Du, X. X., Srour, E. F., Hoffman, R., and Williams, D. A. Interleukin-11 inhibits adipogenesis and stimulates myelopoiesis in human long-term marrow
cultures. Blood, 82: 1428 –1435, 1993.
38. Spiegelman, B. M. PPAR-␥: adipogenic regulator and thiazolidinedione receptor.
Diabetes, 47: 507–514, 1998.
39. Kliewer, S. A., and Willson, T. M. The nuclear receptor PPAR␥– bigger than fat.
Curr. Opin. Genet. Dev., 8: 576 –581, 1998.
40. Fajas, L., Fruchart, J-C., and Auwerx, J. PPAR␥3 mRNA: a distinct PPAR␥ mRNA
subtype transcribed from an independent promoter. FEBS Lett., 438: 55– 60, 1998.
41. Southern, E. M. Detection of specific sequences among DNA fragments separated by
electrophoresis. J. Mol. Biol., 98: 503–517, 1975.
42. Sarraf, P., Mueller, E., Jones, D., King, F. J., DeAngelo, D. J., Partridge, J. B.,
Holden, S. A., Chen, L. B., Singer, S., Fletcher, C., and Spiegelman, B. M. Differentiation and reversal of malignant changes in colon cancer through PPAR␥. Nat.
Med., 4: 1046 –1052, 1998.
43. Mueller, E., Sarraf, P., Tontonoz, P., Evans, R. M., Martin, K. J., Zhang, M., Fletcher,
C., Singer, S., and Spiegelman, B. M. Terminal differentiation of human breast cancer
through PPAR␥. Mol. Cell, 1: 465– 470, 1998.
44. Demetri, G. D., Fletcher, C. D., Mueller, E., Sarraf, P., Naujoks, R., Campbell, N.,
Spiegelman, B. M., and Singer, S. Induction of solid tumor differentiation by the
peroxisome proliferator-activated receptor-␥ ligand troglitazone in patients with liposarcoma. Proc. Natl. Acad. Sci. USA, 96: 3951–3956, 1999.
45. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K. The peroxisome
proliferator-activated receptor-␥ is a negative regulator of macrophage activation.
Nature (Lond.), 391: 79 – 82, 1998.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 2000 American Association for Cancer
Research.
Peroxisome Proliferator-activated Receptor γ Ligands Inhibit
Estrogen Biosynthesis in Human Breast Adipose Tissue:
Possible Implications for Breast Cancer Therapy
Gary L. Rubin, Ying Zhao, Allan M. Kalus, et al.
Cancer Res 2000;60:1604-1608.
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